PROJECT SUMMARY Chronic kidney disease (CKD) affects 14% of Americans, costing 50 billion dollars annually (1). Congenital urinary tract obstruction (CUTO) is the leading cause of CKD in children, and surgical relief of the obstruction does not safeguard against CKD (2, 3). The renal urothelium is an understudied tissue within the kidney, yet early alterations in this tissue have been linked to obstructive nephropathy pathogenesis (4) (5). Preliminary findings reported here identify two mutually exclusive and molecularly distinct cell types that uniquely pattern renal urothelium and dynamically respond to injury. These Krt5+ and Upk+ renal urothelial cells (RUCs) exhibit features unlike those characterized in bladder urothelium. I hypothesize that 1) Krt5+ and Upk+ RUCs pattern renal urothelium in a manner distinct from other classically characterized urothelia; 2) a progenitor-progeny relationship exists whereby Krt5+ RUCs form Upk+ RUCs; and 3) irreversible RUC injury/repair drives the progression of obstructive nephropathy to CKD. To address these hypotheses I propose to: 1) Identify temporal and spatial patterns of embryonic and postnatal RUC development. To date no studies have mapped renal urothelial development at a cellular level. I will identify RUC populations and clarify progenitor- progeny relationships using Cre/LoxP-based fate mapping combined with an innovative focus on dynamic anatomic niches (renal fornix and ureteropelvic junction). 2) Establish diphtheria toxin-mediated cellular depletion as a novel model for urothelial injury. The innovative use of diphtheria toxin-mediated genetic depletion of specific RUC populations will assist me in identifying the roles that these cells play in maintaining urothelial structure and function. 3) Identify the roles of RUCs in injury and repair by indelible labeling and inducible genetic depletion during obstructive nephropathy. The innovative application of fate mapping and diphtheria toxin-mediated genetic depletion in the context of unilateral ureteral obstruction will characterize the dynamic alterations of these cells during obstruction and identify how they ?tip the scale? to CKD progression. Completion of these studies will establish the fundamental knowledge for strategies to augment urothelial regeneration, develop urothelial biomarkers of obstructive injury, and ultimately improve outcomes for children with CUTO.